Gr 1 percp cy5

Single-cell suspensions of tumors were prepared, and flow cytometry was carried out as mentioned earlier [45 (link), 46 (link)]. Cells were stained with the following antibodies obtained from BioLegend: CD16/32 (clone 93, catalog No. 103132), Zombie UV™ Fixable Viability Kit (catalog No. 100752), CD45-PerCP/Cy5.5 (clone 30-F11, catalog No. 103132), CD45-APC/Cy7 (clone 30-F11, catalog No. 103116), CD11b-FITC (clone M1/70, catalog No. 101206), SIRPα-PE (clone BM8, catalog No. 123122), Gr-1-PerCP/Cy5.5 (clone RB6-8C5, catalog No. 108428), F4/80-AF647 (clone N418, catalog No. 117318),CD11c-PE/Cy7 (clone N418, catalog No. 117318), I-A/I-E-AF700 (clone M5/114.15.2, catalog No. 107622), CD86-BV421 (clone GL-1, catalog No. 105032), CD206-BV605 (clone C068C2, catalog No. 141721), CD8a-AF700 (clone 53-6.7, catalog No. 100730), CD8-BV510 (clone 53-6.7, catalog No. 100752), TNF-α-BV421 (clone MP6-XT22, catalog No. 506328), IFN-γ-PE (clone XMG1.2, catalog No. 505808), CD39-PE/Cy7 (clone Duha59, catalog No. 143806), Tim-3-APC/Fire™ 750 (clone RMT3-23, catalog No. 119738), PD-1-BV605 (clone 29 F.1A12, catalog No. 135220). The next antibodies were obtained through eBioscience: Mouse Regulatory T Cell Staining Kit #2 (clone FJK-16s, catalog No. 88-8118-40). FlowJo program (Tree Star Inc.) was utilized to examine the findings that were acquired through a BD LSRFortessa Flow Cytometer.

Lungs were digested as previously described [10 (link)]. Cells were stained with antibodies: CD4-FITC, CD25-APC, FoxP3-PE from e-Bioscience (San Diego, CA, USA), CD4-PerCP-Cy5.5, CD45-APC-Cy7, Gr1-PerCP-Cy5.5, and CD11c-Brilliant Violet 605 from Biolegend (San Diego, CA, USA), CD3-PE-Cy7 and CD8-Alexa Fluor 700 from BD Bioscience (San Diego, CA, USA), CD68-FITC from AbD Serotec (Raleigh, NC, USA) and F4/80 from Life Technologies (Carlsbad, CA, USA). Dead cells were excluded using Live/Dead Fixable Blue Dead Cell Stain kit (Life Technologies, Carlsbad, CA, USA). Cells were analyzed using a LSR II flow cytometer (BD Biosciences, San Diego, CA, USA), and data were analyzed using Flow Jo 7.2 software (Tree Star, Ashland, OR, USA).